Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/30—Partition chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
  • C07K16/065—Purification, fragmentation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/32—Bonded phase chromatography
  • B01D15/325—Reversed phase
  • B01D15/327—Reversed phase with hydrophobic interaction
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
  • B01D15/361—Ion-exchange
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
  • B01D15/3804—Affinity chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
  • B01D15/3804—Affinity chromatography
  • B01D15/3828—Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography

Definitions

• the invention relates to methods of recovering a purified product from a load fluid including one or more impurities.
• the methods comprise passing the load fluid through a medium at operating conditions which cause the medium to bind at least 1 mg of product per mL of medium, and recovering the purified product in the column effluent during the load cycle and any essentially isocratic wash.
• the methods comprise passing the load through a medium at operating conditions defined by a partition coefficient of at least 0.1.
• the desired protein selectively binds to the separation medium and is differentially eluted from the medium by different solvents.
• the impurities specifically bind to the separation medium while the protein of interest does not, thus allowing the recovery of the desired protein in the "flow-through.”
• Current methods for the purification of proteins, such as antibodies include two or more chromatographic steps.
• the first step in the protein purification protocol often involves an affinity chromatography step that utilizes a specific interaction between the protein of interest and an immobilized capture reagent.
• Protein A adsorbents are particularly useful for affinity capture of proteins, such as antibodies, which contain an Fc region.
• drawbacks to using Protein A chromatography for protein purification include leakage of the Protein A capture agent, leading to contamination of the eluted protein product. Additionally, affinity capture does not separate protein variants, such as aggregated forms of the protein, from the protein of interest.
• PCT publication WO 04/076485 describes a method for removing leaked Protein A from an antibody purified by a Protein A chromatography step followed by a flow-through ion exchange step.
• PCT publication WO 03/059935 describes a method for purifying a protein in a sample comprising subjecting the sample to a flow-through hydroxyapatite chromatography step following an affinity chromatography step.
• US Patent 6,177,548 describes a single-step flow-through ion exchange method for removing aggregates from a biological sample where the pH of the sample is adjusted to 0.2 logs below the isoelectric point of the biological sample.
• US Patent 5,451,662 describes a single-step bind-elute ion exchange method where the pH of the crude protein mixture is adjusted to a point between the ranges of isoelectric points of the protein fractions to be separated.
• PCT publication WO 05/044856 describes a single-step displacement method for removal of high molecular weight aggregates from antibody preparations using hydroxyapatite chromatography.
• the present invention relates to methods of recovering a purified product from a load fluid including one or more impurities by passing the load fluid through a medium at operating conditions which cause the medium to bind at least lmg of product per mL of medium and recovering the purified product in the column effluent during the load cycle and any essentially isocratic wash.
• the operating conditions cause the medium to bind at least 5 mg of product per mL of medium.
• the operating conditions cause the medium to bind at least 10 mg of product per mL of medium.
• the operating conditions cause the medium to bind at least 20, 30, 40, 50, or 60 mg of product per mL of medium.
• the present invention also relates to methods of recovering a purified product from a load fluid including one or more impurities by passing the load fluid through a medium at operating conditions defined by a partition coefficient of at least 0.1 and recovering the purified product in the column effluent during the load cycle and any essentially isocratic wash.
• the partition coefficient is in the range of about 0.2 to about 20.0. In another embodiment, the partition coefficient is in the range of about 0.2 to about 10.0. In another embodiment, the partition coefficient is in the range of about 1.0 to about 5.0. In another embodiment, the partition coefficient is in the range of about 0.5 to about 5.0. In an additional embodiment, the partition coefficient is in the range of about 0.5 to about 1.5.
• the present invention also relates to methods of recovering a purified product from a load fluid including one or more impurities by passing the load fluid through a medium at operating conditions which cause the medium to bind from at least 1 to about 70 mg of product per niL of medium and defined by a partition coefficient of 0.3 to 20, and recovering the purified product in the column effluent during the load cycle and any essentially isocratic wash.
• the invention also provides for identifying, in a screening step, the operating conditions that cause the medium to bind at least 1 mg product per mL of medium or alternatively, are defined by a partition coefficient of at least 0.1.
• the screening step can employ batch binding studies or column binding studies, such as gradient elution studies or isocratic elution studies.
• Operating conditions include pH levels, ionic strengths, salt concentrations, excipient concentrations (such as phosphate concentrations, calcium concentrations, arginine concentrations, glycine concentrations, and HEPES concentrations), and counterligand levels
• the medium can be any type of chromatographic resin or separation medium, including a charged ion exchange medium, such as an anion exchange medium or a cation exchange medium, a hydrophobic interaction chromatography resin, a hydroxyapatite resin, or an immobilized metal affinity chromatography resin.
• Purified products that can be recovered using the invention include fusion proteins, Fc-containing proteins, immunoconjugates, cytokines, interleukins, hormones, and therapeutic enzymes.
• Impurities that can be removed using the invention include host cell proteins, nucleic acids, product variants, endotoxins, Protein A, and viruses.
• the medium removes at least 99.9% of the impurities in the load fluid including host cell proteins, nucleic acids, product variants, endotoxins, and
• the concentration of product variants in the purified product is no more than about 2%.
• the load onto the medium may be at a load challenge of at least 500 mg or at least 1000 mg of product per mL of medium.
• a purified product is recovered from a load fluid including one or more impurities by passing the load fluid through a charged ion exchange medium at operating conditions comprising pH levels and ionic strengths which cause the medium to bind at least 1 mg of product per mL of medium or alternatively, at operating conditions defined by a partition coefficient of at least 0.1.
• a purified product is recovered from a load fluid including one or more impurities by passing the load fluid through a hydrophobic interaction chromatography resin at operating conditions comprising pH levels, ionic strengths, and salt concentrations which cause the medium to bind at least 1 mg of product per mL of medium or alternatively, at operating conditions defined by a partition coefficient of at least 0.1.
• a purified product is recovered from a load fluid including one or more impurities by passing the load fluid through a hydroxyapatite chromatography resin at operating conditions comprising pH levels, ionic strengths, phosphate concentrations, calcium concentrations, arginine concentrations, glycine concentrations, HEPES concentrations, and imidazole concentrations which cause the medium to bind at least 1 mg of product per mL of medium or alternatively, at operating conditions defined by a partition coefficient of at least 0.1.
• a purified product is recovered from a load fluid including one or more impurities by passing the load fluid through an immobilized metal affinity chromatography resin at operating conditions comprising counterligand levels and pH levels which cause the medium to bind at least 1 mg of product per mL of medium or alternatively, at operating conditions defined by a partition coefficient of at least 0.1.
• the methods of the invention can be optionally combined with one or more purification steps. The optional step(s) can be performed either prior to or following the practice of the inventive method. For example, the methods of the invention can optionally be combined with a Protein A chromatography step as an initial step.
• a product-containing fluid is eluted from a Protein A column using an elution buffer of low ionic strength; the pH and conductivity of the product-containing fluid is adjusted using a neutralization buffer which results in no more than 2OmM of the ionic strength of the product-containing fluid, resulting in the load fluid; and the load fluid is passed through an anion exchange medium under the operating conditions of the invention.
• the elution buffer comprises molecules with a charged cationic group with a pKa of 6.5-10. In other embodiments, the elution buffer further comprises molecules with a charged anionic group with a pKa of 2-5. In certain embodiments, the elution buffer comprises molecules which are zwitterions at pHs between 7 and 9.
• the invention also provides for purified products, including purified proteins and antibodies, prepared by the methods of the invention.
• Figure 1 shows (A) the relationship between a partition coefficient and a product adsorption isotherm; and (B) adsorption isotherms for product binding to resin, for three modes of operation: bind-elute mode, weak partitioning mode, and flow-through mode.
• Figure 2 shows (A) the partitioning regions for three modes of operation in ion exchange chromatography: bind-elute mode, weak partitioning mode, and flow-through mode; and (B) the partitioning regions for three modes of operation in hydroxyapatite.
• Figure 3 shows schematic chromatograms for three modes of operation: bind- elute mode, weak partitioning mode, and flow-through mode.
• Figure 4 shows a comparison between weak partitioning and flow-through chromatograms.
• Figure 5 shows (A) typical contaminant removal profiles as a function of Kp; and (B) recovery as a function of load challenge and Kp.
• Figure 6 shows typical progression of weak partitioning chromatography step development, including 1) high throughput screen to determine Kp, 2) low load challenge runs, 3) high challenge capacity runs, and 4) optimal weak partitioning chromatography runs.
• Figure 7 shows a contour plot of Kp vs. pH and the total chloride concentration from the low concentration dataset, as described in Experiment 1.1.
• Figure 8 shows Protein A removal as a function of the partition coefficient
• Figure 9 shows a contour plot of logioKp vs. pH and the log of the total chloride concentration, as described in Experiment 2.1.
• Figure 10 shows (A) for Mab-AAB, host cell protein breakthrough profiles as a function of Kp in ion exchange chromatography; and (B) for Mab-AAB, Protein A breakthrough as a function of Kp in ion exchange chromatography.
• Figure 11 shows for Mab-MYA, the optimum operating window for weak partitioning chromatography in hydroxyapatite.
• the optimum Kp in this example is between
• Figure 12 shows for Mab-A5T4, the optimum operating window for weak partitioning chromatography in hydroxyapatite.
• the optimum Kp in this example is between
• Figure 13 shows for Mab-MYO, the optimum operating window for weak partitioning chromatography in hydroxyapatite.
• the optimum Kp in this example is between
• flow-through mode refers to a product preparation separation technique in which at least one product contained in the preparation is intended to flow through a chromatographic resin or medium, while at least one potential contaminant or impurity binds to the chromatographic resin or medium.
• the product partition coefficient for flow-through mode is less than 0.1 and bound product concentration is ⁇ 1 mg/mL.
• the "flow-through mode” is an isocratic operation.
• bind-elute mode refers to a product preparation separation technique in which at least one product contained in the preparation binds to a chromatographic resin or medium.
• the product partition coefficient for bind-elute mode is greater than 20 and the bound product concentrations are between 1 - 20 mg/mL. The bound product in this mode is eluted during the elution phase.
• the term "weak partitioning mode” refers to a product preparation separation technique in which at least one product contained in the preparation, and at least one contaminant or impurity, both bind to a chromatographic resin or medium.
• the binding of product in weak partitioning mode is at least 1 mg of product per niL of chromatographic resin or medium.
• the product partition coefficient for weak partitioning mode is at least 0.1.
• the "weak partitioning mode” is an isocratic operation.
• the term “partition coefficient” (Kp) refers to the equilibrium ratio of the concentration of product absorbed to the resin (Q) to the concentration of product in the solution (c), under specified conditions of pH and solution composition.
• the partition coefficient Kp is also related to the product adsorption isotherms as shown in Figure 1.
• the partition coefficient Kp corresponds to the slope of the product adsorption isotherm at very low solution concentrations. It is related to the maximum capacity as follows:
• Q max is to maximum capacity of the resin for the product
• kd is the dissociation constant for 'resin - product' interaction.
• the partition coefficient is typically measured with a batch binding technique, but other techniques, such as isocratic chromatography, can be used.
• bound product refers to the amount of product which binds to the resin when in equilibrium with a feedstream.
• antibody refers to any immunoglobulin or fragment thereof, and encompasses any polypeptide comprising an antigen-binding site.
• the term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies.
• antibody also includes antibody fragments such as Fab,
• F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function are included in the immunoglobulin family.
• such fragments would comprise an antigen-binding domain.
• the antibody is one which comprises a C H 2/CH3 region and therefore is amenable to purification by Protein A chromatography.
• CH2/CH3 region refers to those amino acid residues in the Fc region of an immunoglobulin molecule which interact with Protein A.
• the C H 2/C H 3 region comprises an intact C H 2 region followed by an intact C H 3 region, and in other embodiments, comprises a Fc region of an immunoglobulin.
• C H 2/C H region-containing proteins include antibodies, immunoadhesions and fusion proteins comprising a protein of interest fused to, or conjugated with, a C H 2/C H 3 region.
• load refers to any load material containing the product, either derived from clarified cell culture or fermentation conditioned medium, or a partially purified intermediate derived from a chromatography step.
• load fluid refers to a liquid containing the load material, for passing through a medium under the operating conditions of the invention.
• impurity refers to any foreign or objectionable molecule, including a biological macromolecule such as a DNA, an RNA, or a protein, other than the protein of interest being purified that is also present in a sample of the protein of interest being purified.
• Impurities include, for example, protein variants, such as aggregated proteins, high molecular weight species, low molecular weight species and fragments, and deamidated species; other proteins from host cells that secrete the protein being purified (host cell proteins); proteins that are part of an absorbent used for affinity chromatography that may leach into a sample during prior purification steps, such as Protein A; endotoxins; and viruses.
• protein variants such as aggregated proteins, high molecular weight species, low molecular weight species and fragments, and deamidated species
• other proteins from host cells that secrete the protein being purified host cell proteins
• proteins that are part of an absorbent used for affinity chromatography that may leach into a sample during prior purification steps, such as Protein A; endotoxins; and viruses.
• essentially isocratic wash refers to a solution which varies only slightly from the load fluid in terms of composition or pH.
• column effluent refers to the liquid exiting the medium or column during the load cycle, or in the period that the load is being applied.
• load challenge refers to the total mass of product loaded onto the column in the load cycle of a chromatography step or applied to the resin in batch binding, measured in units of mass of product per unit volume of resin.
• log removal value refers to the log(base 10) of the ratio of the mass of impurity in the load of a purification step to the mass of impurity in the product pool.
• isocratic chromatography refers to the operation of a chromatographic column with a solvent that does not change strength during the period of interest.
• the present invention provides methods for recovering purified products from a load fluid containing one or more impurities.
• the invention has application to the large- scale preparation of proteins for therapeutic and diagnostic purposes.
• weak partitioning mode a load fluid containing a product of interest and one or more impurities is passed through a chromatographic medium, with both the product and the impurities binding to the medium. However, the impurities bind more tightly to the medium than the product and as loading continues, unbound product passes through the medium and is recovered from the column effluent.
• the medium is optionally subsequently washed under isocratic conditions to recover additional weakly bound product from the medium and the purified product from any essentially isocratic wash is pooled with the purified product from the column effluent during the load cycle.
• weak partitioning mode is defined by operating conditions which cause the medium to bind at least 1 mg of product per mL of medium.
• the operating conditions cause the medium to bind at least 5 mg of product per mL of medium. In another embodiment, the operating conditions cause the medium to bind at least 10 mg of product per mL of medium. In another embodiment, the operating conditions cause the medium to bind at least 20 mg of product per mL of medium. [0062] In certain embodiments of the invention, the total product mass bound to the medium is at least 10% of the total product mass loaded onto the medium. In some embodiments, the total product mass bound to the medium is at least 20% of the total product mass loaded onto the medium. In other embodiments, the total product mass bound to the medium is at least 30% of the total product mass loaded onto the medium.
• weak partitioning mode is also defined by a partition coefficient of at least 0.1.
• operating in weak partitioning mode comprises operating under conditions defined by a partition coefficient in the range of about 0.2 to about 20.0.
• operating in weak partitioning mode comprises operating under conditions defined by a partition coefficient in the range of about 0.2 to about 10.0.
• operating in weak partitioning mode comprises operating under conditions defined by a partition coefficient in the range of about 1.0 to about 5.0.
• operating in weak partitioning mode comprises operating under conditions defined by a partition coefficient in the range of about 0.5 to about 5.0.
• operating in weak partitioning mode comprises operating under conditions defined by a partition coefficient in the range of about 0.5 to about 1.5.
• At least one embodiment of the present invention provides weak partitioning mode operating conditions which cause the medium to bind from at least 1 to about 70 mg of product per mL of medium, and which are defined by a partition coefficient of 0.3 to 20.
• Figure 1 shows the product adsorption isotherms for the bind-elute, flow- through, and weak partitioning modes, with product binding for weak partitioning mode being clearly intermediate in comparison to bind-elute and flow-through modes.
• FIG. 2A depicts the partitioning regions for bind-elute, weak partitioning, and flow-through modes as a function of ionic strength, showing that Ki mp is higher in weak partitioning mode than in flow-through mode.
• Table A summarizes the differences in characteristics between the three modes of binding: bind-elute (B-E), weak partitioning (WP), and flow-through (FT).
• Weak partitioning mode can also be distinguished from bind-elute and flow- through modes by their chromatograms, as shown in Figure 3.
• the chromatograms for flow-through and weak partitioning modes may seem quite similar - the product is recovered in the column effluent and wash fractions, under isocratic conditions.
• subtle, but meaningful distinctions exist in the chromatograms which can be used to distinguish these modes, as shown in Figure 4.
• a small strip peak containing product may be present (which corresponds to the resin still binding 10 - 50% of the load product concentration after the wash stage), which can be recovered from the resin by applying a 1 - 5 CV wash after the load under isocratic conditions subsequent to recovery of the column effluent during the load cycle.
• Figure 5A shows the general trends in contaminant LRV for various levels of product partition coefficient values.
• Contaminant LRVs are relatively low at Kp conditions corresponding to flow-through operations. Operating under conditions of increasing Kp significantly increases LRVs in the column effluent fractions prior to contaminant breakthrough. As shown in the examples, operating at higher Kp values improves the contaminant LRVs by as much as 2 logs from those corresponding to the standard flow- through conditions.
• the upper Kp limit for weak partitioning chromatography is also dependent on the column load challenge as shown in Figure 5B.
• the partition coefficient has no impact on product recovery at values bordering flow-through conditions.
• the product recovery begins to drop at high Kp values where the isocratic wash conditions are is not effective at washing the bound product off the column in a reasonable number of wash volumes.
• the extent of product loss due to ineffective washout is sensitive to load challenge, as well as the nature and proportion of contaminant in the load.
• the lower limit of the WP region is defined by requirements of contaminant removal, while the upper limit for a given load challenge is defined by constraints of product recovery or capacity.
• optimal weak partitioning conditions may be identified using the following sequence of experiments, as shown in Figure 6:
• Weak partitioning mode may be used in conjunction with any chromatographic resin or medium for separation of a product from impurities.
• the medium is a charged ion exchange medium.
• Ion exchange is a form of chromatography that separates according to net charge. Separation of molecules occurs as a result of the competition between the charged product of interest and counterions for oppositely charged ligand groups on the ion exchange medium. Binding interactions between the product and an ion exchange medium depend on the net charge of the product. Net charge is dependent on the pH and ionic strength of the medium, which affects the different charge characteristics of amino acids and other components on the exposed surface of the product molecule(s) of interest.
• Ion exchange resins that may be used in the invention include anion exchange resins and cation exchange resins.
• Anionic exchange resins may employ substituents such as diethylaminoethyl (DEAE), trimethyalaminoethyl (TMAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups.
• Cationic exchange may employ substituents such as carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).
• Cellulosic ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K. Sephadex-based and cross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-Sephadex, and DEAE-, Q-, CM- and S-Sepharose, and Sepharose are all available from Amersham Biosciences, Piscataway, NJ.
• DEAE and CM derivatized ethylene glycol- methacrylate copolymer such as TOYOPEARLTM DEAE-650S or M and TOYOPEARLTM CM-650S or M are available from Toso Haas Co., Philadelphia, PA.
• weak partitioning mode is used with a hydrophobic interaction chromatography (HIC) resin for product purification.
• HIC is a technique for separating molecules based on hydrophobicity.
• sample molecules in a high salt buffer are loaded onto the HIC resin.
• the salt in the buffer interacts with water molecules to reduce the solvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules which are consequently absorbed by the HIC medium.
• the more hydrophobic the molecule the less salt needed to promote binding. Binding interactions between the product molecules and a HIC medium thus depend on conditions such as pH, ionic strength, and salt concentrations of the medium.
• HIC resins that can be used in the invention include resins comprising a base matrix (e.g., cross-linked agarose or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled.
• a base matrix e.g., cross-linked agarose or synthetic copolymer material
• hydrophobic ligands e.g., alkyl or aryl groups
• examples include Phenyl SEPHAROSETM 6 FAST FLOWTM (Pharmacia LKB Biotechnology, AB, Sweden); Phenyl SEPHAROSETM High Performance (Pharmacia LKB Biotechnology, AB, Sweden); Octyl SEPHAROSETM High Performance (Pharmacia LKB Biotechnology, AB, Sweden); FractogelTM EMD Propyl or FRACTOGELTM EMD Phenyl (E.
• MACRO-PREPTM Methyl or MACRO-PREPTM t-Butyl Supports Bio-Rad, CA
• WP HI- Propyl (C 3 )TM J. T. Baker, NJ
• TOYOPEARLTM ether, phenyl or butyl TOsoHaas, PA.
• weak partitioning mode is used with hydroxyapatite chromatography for product purification. Hydroxyapatite chromatography is a technique that utilizes an insoluble hydroxylated calcium phosphate of the formula [Ca 1C )(PO 4 ⁇ (OH) 2 ], as both the matrix and the ligand.
• Functional groups consist of pairs of positively charged calcium ions (C-sites) and clusters of negatively charged phosphate groups (P-sites). Binding interactions between the product and the hydroxyapatite medium depend on conditions such as the pH, ionic strength, and excipient concentrations, such as phosphate concentrations, calcium concentrations, arginine concentrations, glycine concentrations, and HEPES concentrations of the medium.
• Various hydroxyapatite chromatographic resins are available commercially and can be used in the invention.
• weak partitioning mode is used with an immobilized metal affinity chromatography (JJVIAC) resin for product purification.
• JJVIAC is based on the interaction between chelated transition metal ions immobilized on the resin and the imidazole side chains of histidine residues on the tagged product of interest. Separation of molecules occurs as a result of competition between the tagged product of interest and counterligands for metal groups on the MAC resin. Binding interactions between the product and metal-charged IMAC medium depend on conditions such as counterligand levels, such as imidazole concentrations, and ionic strength of the medium.
• IMAC resins are available commercially and can be used in the invention.
• the invention can be used for the commercial-scale purification of various products of interest, including naturally occurring proteins, fusion proteins, Fc-containing proteins, immunoconjugates, cytokines, interleukins, hormones, and therapeutic enzymes.
• the protein undergoing purification may comprise one or more constant antibody immunoglobulin domain(s).
• the protein may also comprise a single or multiple variable antibody immunoglobulin domain(s).
• the Fc-containing protein may comprise an antibody.
• the proteins can be derived from various sources, including cultured recombinant prokaryotic or eukaryotic host cell lines.
• the antibody preparations of the invention can be isolated from a number of sources including, but not limited to, serum of immunized animals, ascites fluid, hybridoma or myeloma supernatants, conditioned media derived from culturing a recombinant cell line that expresses the antibody molecule and from all cell extracts of antibody-producing cells.
• antibodies from conditioned cell culture media of a variety of antibody producing recombinant cell lines are purified. Although one may expect some variation from cell line to cell line and among the various antibody products, based on the disclosure herein, it is well within the purview of one of ordinary skill in this art to adapt the invention herein to a particular combination of antibody protein and producing cell line.
• this invention was applied to the purification of several antibodies of the IgG isotype. More specifically, this invention was applied to purification of a humanized, anti-A beta monoclonal antibody, an anti-GDF8 antibody, and a humanized, anti-IL-13 monoclonal antibody.
• the region of weak partitioning Before loading the fluid containing the product and impurities onto the medium, it may be necessary to identify the region of weak partitioning by finding the operating conditions which cause the medium to bind at least 1 mg of product per mL of medium. In one embodiment, the operating conditions found cause the medium to bind at least 5 mg of product per mL of medium. In another embodiment, the operating conditions found cause the medium to bind at least 10 mg of product per mL of medium. In other embodiments, the operating conditions found cause the medium to bind at least 20 mg of product per mL of medium.
• the weak partitioning region is identified by finding the operating conditions defined by a partition coefficient of at least 0.1.
• the operating conditions found are defined by a partition coefficient in the range of about 0.2 to about 20.0. In other embodiments, the operating conditions found are defined by a partition coefficient in the range of about 0.2 to about 10.0. In yet other embodiments, the operating conditions found are defined by a partition coefficient in the range of about 1.0 to about 5.0, in the range of about 0.5 to about 5.0, or in the range of about 0.5 to about 1.5.
• the weak partitioning region is identified by finding the operating conditions which cause the medium to bind from at least 1 to about 70 mg of product per mL of medium and which is defined by a partition coefficient of 0.3 to 20.
• the appropriate operating conditions will depend on the choice of medium selected for purification of the product.
• the operating conditions comprise pH levels and ionic strengths.
• the operating conditions further comprise salt concentrations.
• the operating conditions further comprise excipient levels, such as phosphate concentrations and calcium concentrations.
• the operating conditions comprise counterligand levels, such as imidazole concentrations, and pH levels.
• a screening step can be used to identify the operating conditions for weak partitioning mode. Such a screening step could include batch binding studies or column binding studies. Column binding studies could include gradient elution studies or isocratic elution studies.
• one skilled in the art can determine which buffer or salt is appropriate for the particular protein being purified and for the operating conditions that are being identified.
• the optimal concentration of the selected buffer or salt can then be determined by, for example, running a gradient of the selected buffer or salt through a column to which a load fluid comprising the product to be purified and the impurities has been applied. Fractions of the effluent of the column can be collected and analyzed to determine the concentration of buffer or salt at which product binding is at least 1 mg of product per mL of medium or alternatively, at which the partition coefficient for the product is at least 0.1.
• the partition coefficient is measured between 1 and 10 mg/mL load challenge with a phase ratio (volume of liquid to volume of resin) of three to six in a batch binding experiment.
• the conditions of the load fluid and/or medium can be adjusted accordingly.
• the medium can be equilibrated by washing it with a solution that will bring it to the necessary operating conditions of weak partitioning mode.
• the load fluid may also be buffer exchanged into an appropriate buffer or load buffer in preparation for weak partitioning mode.
• the load buffer can be the same or a different buffer as the equilibration buffer.
• the ionic strength of the load fluid is no more than 100 mM. In another embodiment, the ionic strength of the load fluid is no more than 50 mM. In another embodiment, the ionic strength of the load fluid is no more than 25 mM. In yet another embodiment, the ionic strength of the load fluid is no more than 10 mM.
• the load fluid may be passed through a separation medium that is packed in a bed column, packed in a fluidized/expanded bed column containing the solid phase matrix, and/or in a batch format where the solid phase matrix is mixed with the load fluid for a certain time.
• the medium is optionally washed with a volume of essentially isocratic wash.
• Purified product can be obtained from any essentially isocratic wash and pooled with the purified product from the column effluent during the load cycle.
• the medium can optionally be stripped and regenerated. This procedure is typically performed regularly to minimize the buildup of impurities on the surface of the solid phase and/or to sterilize the medium to avoid contamination of the product with microorganisms.
• the concentration of product in the load fluid is at least 1 mg of product per mL of load fluid, in another embodiment, the concentration of product in the load fluid is at least 5 mg of product per mL of load fluid, in another embodiment, at least 50 mg of product per mL of load fluid, and in another embodiment, at least 100 mg of product per mL of load fluid.
• Purified product can be recovered from up to 50 CVs of load fluid passed through the medium.
• the load onto the medium may be at a load challenge of at least 10 mg of product per mL of medium. In other embodiments, the loading of the product onto the medium is at least 50 mg of product per mL of medium. It certain embodiments, the loading of the product onto the medium is at least 100 mg of product per mL of medium. In other embodiments, the load onto the medium may be at a load challenge of at least 500 mg of product per mL of medium. In yet other embodiments, the load onto the medium may be at a load challenge of at least 1000 mg of product per mL of medium. 5. Removal of impurities
• Weak partitioning mode has been shown to be useful for removing all types of impurities from product preparations, including host cell proteins, nucleic acids, product variants, including aggregated product and high molecular weight species, endotoxins, viruses, and Protein A contaminants from prior purification steps.
• the concentration of host cell proteins present in the purified product is no more than about 500 ng host cell proteins per mg of product. In other embodiments, the concentration of host cell proteins can be reduced to no more than 250 ng per mg of product, and in other embodiments, to no more than 100 ng per mg of product. In certain embodiments, the log removal value of host cell proteins is at least 1.0, in other embodiments, the log removal value of host cell proteins is at least 2.0, and in other embodiments, the log removal value of host cell proteins is at least 3.0. [0093] In one embodiment of the invention, the concentration of Protein A present in the purified product is no more than about 100 ng Protein A per mg of product.
• the concentration of Protein A can be reduced to no more than 50 ng per mg of product, and in other embodiments, to no more than 10 ng per mg of product.
• the log removal value of Protein A is at least 1.0, and in other embodiments, the log removal value of Protein A is at least 2.0, and in other embodiments, the log removal value of Protein A is at least 3.0.
• viral impurities are removed from the purified product.
• the log removal value of viruses is greater than 1.0, in other embodiments, greater than 2.0, and in other embodiments, greater than 3.0.
• nucleic acid impurities are removed from the purified product.
• the amount of nucleic acids present in the purified product can be reduced to no more than 1 ng nucleic acids per mg of product.
• the concentration of protein variants in the purified product is no more than about 10%.
• the concentration of protein variants can be reduced to no more than about 5%, in some embodiments, to no more than 2%, and in some embodiments, to no more than 0.5%.
• the separation medium removes at least 90% of host cell protein, nucleic acid, protein variant, endotoxin, and Protein A impurities. In some embodiments, the medium removes at least 99% of the impurities, and in other embodiments, the medium removes at least 99.9% of the impurities.
• the purification method of the invention can be used in combination with other protein purification steps.
• one or more steps preceding the weak partitioning step may be desirable to reduce the load challenge.
• one or more purification steps following the weak partitioning step may be desirable to remove additional contaminants or impurities.
• the weak partitioning purification procedure described may optionally be combined with other purification steps, including but not limited to, Protein A chromatography, affinity chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, size exclusion chromatography, diafiltration, ultrafiltration, viral removal filtration, and/or ion exchange chromatography.
• the harvest media may optionally be initially purified by a Protein A chromatography step.
• PROSEP- ATM which consists of Protein A covalently coupled to controlled pore glass, can be employed.
• Protein A Sepharose FAST FLOWTM (Amersham Biosciences, Piscataway, NJ)
• TOYOPEARLTM 650M Protein A TosoHaas Co., Philadelphia, PA
• MABSELECTTM columns (Amersham Biosciences, Piscataway, NJ).
• a product-containing fluid is eluted from a Protein A column using an elution buffer of low ionic strength.
• the pH and conductivity of the product-containing fluid is then adjusted using a neutralization buffer, which results in no more than 2OmM of the ionic strength of the product-containing fluid.
• the resulting load fluid is then passed through an anion exchange medium or hydroxyapatite medium operating under conditions of weak partitioning mode.
• the load fluid is passed through an anion exchange medium without the need for diafiltration.
• the pH and conductivity of the product-containing fluid is adjusted using a neutralization buffer which results in no more than 4OmM of the ionic strength of the product-containing fluid.
• the pH and conductivity of the product-containing fluid is adjusted using a neutralization buffer that results in no more than 6OmM of the ionic strength of the product-containing fluid. In yet other embodiments, the pH and conductivity of the product-containing fluid is adjusted using a neutralization buffer that results in no more than 8OmM of the ionic strength of the product-containing fluid.
• Buffers that can be used for elution from the Protein A column include buffers comprising molecules with a charged anionic group with a pKa of 2-5. Such elution buffers could further comprise molecules with a charged cationic group with a pKa of 6.5-10.
• the elution buffer comprises molecules which are zwitterions at pHs between 4 and 9, such as glycine; l,4-piperazinebis-(ethanesulfonic acid); glycylglycine; cyclopentanetetra-1 ,2,3,4-carboxylic acid; N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; 2-(N-morpholino)propane-sulfonic acid; N-tris(hydroxylmethyl)methyl-2-aminoethane sulfonic acid; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 4-(2-hydroxyethyl)-I- piperazinepropane sulfonic acid; N-tris(hydroxymethyl)methylglycine; glycinamide; N,N- bis(2-hydroxyethyl)glycine; N-tris(hydroxymethyl)methyl-2-amino
• the elution of a Protein A column with a zwitterionic buffer provides the advantage of low ionic strength upon some degree of neutralization.
• the low ionic strength of the buffer does not adversely impact the operation of subsequent ion exchange columns, including hydroxyapatite columns.
• High levels of ionic strength will decrease the binding of impurities to ion exchange columns, which may decrease the overall efficiency of the purification.
• Lower ionic strength solutions are preferred for loads onto ion exchange columns, as the ionic strength can be raised easily with the addition of concentrated salt solutions; decreasing the ionic strength of solutions is not facile.
• buffers that have a low pKa that allow use at low pH levels useful in Protein A elution steps, but that also have a second pKa that allow use at higher pH levels useful in ion exchange chromatography; these buffers, if used at a proper second pH, have little effective charge during the operation of the ion exchange step subsequent to the Protein A step.
• a zwitterionic buffer that has a pKa near that of the elution pH preferred for
• Protein A (between pH 2 and 5, preferably between 2.5 and 4.0) allows the buffer to be used to maintain the pH near the buffer's pi and to elute the column.
• Zwitterionic buffers that also have a pKa near that of the operation of a subsequent ion exchange column (pH 5.5 to 11) would allow the buffer to control the pH in this pH range as well as in the Protein A elution pH range.
• a zwitterionic buffer with pKal and pKa2 can elute a Protein A column at pH levels within one pH unit of pKal . Further, if the Protein A pool is neutralized with a basic solution of the zwitterionic buffer to a second pH within one pH unit of pKa2, the zwitterionic buffer will be able to maintain the pH of the solution. If the second pH is below that of pKa2, the buffer remains zwitterionic and contributes little to the overall ionic strength of the solution. For example, a zwitterionic buffer with concentration x at a pH equal to the pKa2 will contribute to the overall ionic strength only x/2.
• a zwitterionic buffer with concentration x at 1 pH unit below the pKa2 of the buffer will have a ionic strength of one-tenth of x. This reduction in ionic strength is significantly useful for operation of ionic exchange chromatography.
• the existence of buffers that have a pKal useful for elution of Protein A columns and a pKa2 useful for operation of ion exchange chromatography is not obvious, in that the pKal of these buffers is not commonly reported. Buffers are commonly used at pH levels near that of pKa2, and are not known by those skilled in the art of chromatography to be useful for elution from a Protein A column.
• these buffers for elution from Protein A chromatographic columns is not generally realized, it is also not realized that these zwitterionic buffers would have additional utility as buffers for ion exchange columns subsequent to Protein A columns because they contribute less ionic strength to the neutralized Protein A pool.
• Examples are provided for three modes of chromatography (anion exchange, hydrophobic interaction, and hydroxyapatite), using three different monoclonal antibodies.
• Four separate series of experiments are described, each representing a different pairing of the chromatography mode and the monoclonal antibody to be purified.
• the initial screening studies are presented first, which determine the partition coefficient and/or the concentration of product bound to the resin under various solution conditions, thus defining the operating regions of weak partitioning (WP) and flow-through (FT) modes.
• WP weak partitioning
• FT flow-through
• ELISA Protein A enzyme-linked immunosorbent assay
• SEC analytical size exclusion chromatography
• HCPs host cell proteins
• a high throughput screen was performed to identify the weak partitioning and flow-through conditions for Mab-AAB with TMAE-HiCap (M) medium.
• This screen varied the concentration of sodium chloride and pH to determine their effect on the extent of binding of MAB-AAB and process related impurities (Protein A and HCP) to the TMAE medium.
• TMAE HiCap medium 50 ⁇ L of TMAE HiCap medium was added to each well of a 96 well filter plate. Each well was equilibrated in solutions made up 5OmM glycine and a variable amount of Tris buffer (depending upon the amount needed for neutralization to the pH specified in Table 1.1.1) and sodium chloride (specified in Table 1.1.2). The pH ranged from 7.6 to 9.0 and the sodium chloride ranged from OmM to 8OmM.
• the buffer solutions used in each row were diluted on an automated pipetting system (Tecan 100 RST).
• the stock solution for the buffers were made from 50OmM glycine acidified with HCl to pH 3.0, and subsequently neutralized with 2M Tris Base to the pH levels indicated in Table 1.1.1. This titration resulted in a level of Tris that depended upon the pH of the buffer.
• the buffer pH was measured at a 1 to 10 dilution of the stock buffer concentration, which corresponded to the dilution made by the automated pipetting system.
• the buffer contributes about 1OmM of ionic strength to the final solution.
• Table 1.1.2 NaCI levels (in mM) and protein challenges (mg/mL) in each well
• each well was equilibrated in the conditions of NaCl and pH as described in Tables 1.1.1 and 1.1.2 in a phase volume ratio of 6:1 (30OuL solution: 5OuL resin). The plate was shaken for 20 minutes, allowing equilibrium to be reached. The solution was then removed by centrifuging the filter plate. This equilibration cycle was repeated three times.
• MAb-AAB solution to 5 mg/mL of resin with a volume ratio of 6: 1 (30OuL solution: 5OuL resin) at the appropriate NaCl concentration and pH.
• a 36mg/mL solution of Mab-AAB in ImM HEPES, 1OmM NaCl, pH 7.0 spiked with 300ppm of Protein A was used as stock solution.
• the loaded plate was shaken for 20 minutes, allowing the resin and solution to equilibrate.
• the supernatant was removed from the filter plate by centrifugation and collected into a collection plate.
• the protein concentration in the supernatant in each well was determined by absorbance at A280nm.
• the Kp value can be used to describe regions where
• MAB-AAB binds to the TMAE medium with different strengths. These regions are more clearly visualized in Figure 7.
• K ⁇ 0.1
• K ⁇ 0.1
• K 0.1
• K ⁇ K ⁇ 20 weak partitioning
• Table 1.1.4 There is a region of pH and conductivity where the TMAE chromatography step provides very significant removal of Protein A with limited protein loss to the resin. This region was found to be closely correlated to the partition coefficient value, Kp, and not any specific pH or chloride concentration (see Figure 8).
• the culture containing the monoclonal antibody was purified at Pilot scale using a MabSelect column (2,389 mL) connected to a Millipore K-prime 400 chromatography system.
• a Mabselect Protein A column was equilibrated with 5 column volumes of 50 mM Tris/150 mM NaCl, pH 7.5 at a flow rate of 300 cm/hr. The column was then loaded at a load of approximately 40 mg product/ml resin. This was followed by a 5CV wash in IM NaCl, 5OmM Tris, pH 7.5 and a 5CV wash containing 10 mM Tris, 75mM NaCl, pH 7.5 wash.
• the column was then eluted using 50 mM glycine, 75mM NaCl, pH 3.0.
• the product pool was neutralized to pH 7.6 using 2M Tris pH 8.5.
• the neutralized peak had a chloride concentration of approximately 9OmM.
• the neutralized Protein A pool was further purified over the TMAE step with the equilibration, load, and wash solutions at pH 7.5 with 50 mM Tris and 75 mM sodium chloride. 5 column volumes of wash were used.
• the column dimensions and load challenges for these two studies were: Run 1: 7.0 cm diameter x 20.6 cm bed height (volume - 793 mL) with a load concentration of 11.9 mg/mL, and Run 2: 7.0 cm diameter x 13 cm bed height (volume - 500 mL) with a load concentration of 17.6 mg/mL.
• the impurities in the load were 25,398 ppm of HCP, 99.5 ppm of Protein A, and 2.3% HMW.
• + includes the Cl- ion contribution from NaCl, buffers and titrants
• a high throughput screen was performed to identify the weak partitioning and flow -through conditions for Mab-IMA with TMAE-HiCap (M) medium.
• This screen varied the concentration of sodium chloride and pH to determine their effect on the extent of binding of MAB-IMA and process related impurities (Protein A and HCP) to the TMAE medium.
• lOO ⁇ L of TMAE HiCap medium was added to each well of a 96 well filter plate. Each well was equilibrated in solutions made up of 25mM buffer no more than 1 pH unit away from the buffer pKa (Table 2.1.1) and the appropriate level of sodium chloride (Table 2.1.2).
• AU buffers were titrated to the target pH using 12M HCl. As a result of different buffering species required different levels of titrant, the chloride concentration varied from well to well depending on which buffer was used for that well.
• the amount of Cl- needed to titrate the buffer to the target pH was calculated using the Henderson- Hasselbach equation and added to the total Cl- contributed from both the NaCl and the amount in the load material. The calculated Cl- level for each well in the experiment is listed in Table 2.1.3.
• each well was equilibrated in the conditions of NaCl and pH as described in Tables 2.1.1 and 2.1.2 in a phase volume ratio of 3:1 (30OuL solution: lOOuL resin). The plate was shaken for 20 minutes, allowing equilibrium to be reached. The solution was then removed by centrifuging the filter plate. This equilibration cycle was repeated three times.
• MAb-IMA solution to 3 mg/mL of resin with a volume ratio of 3:1 (30OuL solution: lOOuL resin) at the appropriate NaCl concentration and pH.
• a 30mg/mL solution of Mab-IMA in ImM Mes, 15mM NaCl, pH 6.5 with 300ppm of Protein A was used as stock solution.
• the loaded plate was shaken for 20 minutes, allowing the resin and solution to equilibrate.
• the supernatant was removed from the filter plate by centrifugation and collected into a collection plate.
• the protein concentration in the supernatant in each well was determined by absorbance at A280nm. Any decrease in Protein A and/or HCP levels indicates a condition conducive to purification.
• the Kp value can be used to describe regions where
• MAB-EVIA binds to the TMAE medium with different strengths. These regions are more clearly visualized in Figure 9.
• the strength of MAb-EvIA binding to TMAE medium can be manipulated by varying conditions of pH and chloride concentration into flow-through (Kp ⁇ O.l), weak partitioning (0.1 ⁇ Kp ⁇ 20), and binding zones (Kp>20).
• the supernatant from the load stage of several wells from each zone were sampled and submitted for Protein A analysis. The load had 300 ppm of Protein A.
• the assay results of these samples are summarized in Table 2.1.5.
• Predicted Kp values are derived from a response surface fit to the HTS screen, and subsequent prediction of the Kp based on this regression model.
• the bound product was determined by measuring the protein in the column strip using UV absorbance. This method of determining the amount of bound product typically underestimates the total amount of product bound due to isocratic elution of product in the wash. Protein A, HCP, HMW and LMW removal results from these experiments are also presented in Table 2.2.1. There is relatively poor and variable removal of HCP, and no removal of Protein A and product variants (HMW and LMW species).
• the TMAE process step for the purification of Mab-MA operated in the weak partitioning zone was scaled-up to the Pilot plant and clinical manufacturing.
• the culture containing the monoclonal antibody was first purified using a 3L or 5L MabSelect column in Pilot and a 28 L MabSelect column during clinical manufacturing.
• the MabSelect column was essentially operated as described in Experiment 1.2.
• the neutralized Protein A peak pools from these runs were further purified on a 1.5 L TMAE column in Pilot and a 7 L TMAE column in the clinical manufacturing facility.
• Tables 2.4.1 and 2.4.2 The results of three Pilot runs and nine clinical manufacturing runs are summarized in Tables 2.4.1 and 2.4.2, respectively.
• step performance was consistent across the runs, with excellent reduction of HCP, Protein A, and good removal of product related HMW and LMW species.
• Product recovery was >87% in all runs.
• An estimate of the product bound to the resin during the Pilot runs was obtained from the product in the column strip, which ranged from 6 - 14 mg/mL of resin.
• HTS identified conditions for WP and FT operation.
• the FT mode provided only a modest reduction in HCP and LMW species, and no reduction in Protein A residuals or HMW species. Operation in the WP mode improves the removal of all impurities without sacrificing product yield.
• the process step was scaled up to the Pilot plant and operated consistently for three runs, with very high LRVs for HCP and Protein A, and good reductions in HMW and LMW species.
• the column was equilibrated with 5 column volumes of equilibration buffer 1 followed by 5 column volumes of equilibration 2 step. The column was then loaded to between 940 and 2144 mg of product /ml of resin with the Protein A peak pool (refer to Series 1, Experiment 1.1) adjusted to the appropriate equilibration 2 buffer. [0152] The column effluent fractions were collected and subsequently assayed for
• the product bound value for the ran corresponding to a Kp of 0.1 was near zero, as is expected for a typical flow-through operation.
• the product bound values for experiments performed in the weak partitioning region were > 12.0 mg/ml in all cases.
• the product bound value for the run corresponding to the Kp of 7 was as high as 71 mg/ml.
• the product recovery in the combined load eluate and wash fractions in all cases were, however, > 93%.
• FIGS. 1OA and 1OB the HCP removal increases significantly as conditions move from flow-through to weak partitioning. Operating under flow-through conditions provides approximately 1.5 logs of HCP clearance, while the HCP log removal values were as high as 3.8 logs at load challenges ⁇ 450 mg /ml of resin when operated at a Kp of 7 in the weak partitioning region. At a Kp of 0.8 in the weak partitioning region, 2.8 logs of HCP clearance was obtained for load challenges up to 1000 mg/ml of the resin, and > 3 logs of HCP clearance was obtained for up to a load challenge of 800 mg/ml of resin at a Kp of 2.7 in the weak partitioning region.
• Mab-AAB which was partially purified by Protein A chromatography was diluted into Tris/ Na 2 SO 4 solution to a final of concentration of 0.87 mg/ml. 50 ul of resin was equilibrated with 300 ul of buffer and then the supernatant decanted for each of the Tris/ Na 2 SO 4 conditions; this equilibration was repeated three times. After equilibration, decanted resin was mixed with product at the same salt concentration and pH and incubated for 30 minutes with gentle shaking. The load challenge was 5.2 mg product /ml of resin for all conditions. A UV plate was then stacked at the bottom of the filter plate to collect the supernatant upon centrifugation.
• Table 4.2.1 summarizes the partition coefficients from this experiment. The highest concentrations of Na 2 SO 4 caused strong product binding, while salt concentrations in the range of 0.40 - 0.55M represent weak partitioning conditions.
• Protein A step run essentially the same as those previously described or ii) more pure TMAE Q Sepharose FF product pools from FT mode operation.
• the runs are ranked by the partition coefficients.
• the bound product was determined by measuring the protein in the column strip using UV absorbance. This method of determining the bound product typically underestimates the amount of product bound during the load due to the gradual desorption of the product during the wash.
• HIC step improves significantly with respect to contaminant reduction as we move from flow-through conditions to weak partitioning conditions, while product recovery is comparable.
• a further increase in the operating salt concentration leads to partition coefficients that correspond to the strong binding conditions.
• the optimum operating window for this separation therefore corresponds to that of weak partitioning chromatography.
• the HIC step provides 1 log reduction of HCP and a 3.4 fold reduction of Protein A.
• the Q Sepharose FF peak pool was used in these sets of experiments to highlight the fact that the performance of the HIC step under the optimum weak partitioning chromatography conditions can be further improved with a cleaner feedstock.
• the load material in this case contained 2880 ppm of HCP and was generated by purifying the Protein A peak pool on a Q-Sepharose FF column.
• the Q-Sepharose peak was diluted to 3.27 mg/ml at 550 mM Na 2 SO 4 and loaded to the column to a load challenge of 100 mg/ml of resin for operation under weak partitioning conditions.
• the column was subsequently washed with 10 CVs of a buffer containing the same salt concentration as the load and stripped with 6CV of 50 mM Tris buffer, pH 7.5.
• the second experiment was conducted under flow-through conditions. The load was adjusted to 3.03 mg/ml in 200 Na 2 SO 4 and loaded to the column to a load challenge of 90 mg/ml of resin. The column was then washed with 6CY of a buffer containing the same salt concentration as the load, and subsequently stripped with 6 CV of 50 mM Tris buffer, pH 7.5. In both runs, the flow- through and wash fractions were collected for recovery and impurity analysis. The results from these runs are reported in Table 4.2.2.
• HCP LRV across the HIC step with either feedstock was comparable under flow-through conditions ( ⁇ 0.4 - 0.5 LRV).
• HCP LRV values for experiments performed under weak partitioning conditions increased from 1 LRV with the Protein A load material to greater than 2 LRV with load material purified through Protein A and Q
• HIC chromatography
• a high throughput screen was performed to identify the weak partitioning and flow-through conditions for Mab-MYA with ceramic hydroxyapatite medium. This screen varied the concentration of sodium chloride and sodium phosphate to determine their effect on the extent of binding of MAB-MYA to the hydroxyapatite medium.
• 50 ⁇ L of ceramic hydroxyapatite medium was added to 30 wells of a 96 well filter plate. Each well was equilibrated in solutions made up of the appropriate sodium chloride and sodium phosphate concentrations in a 10OmM HEPES buffer containing 10OmM arginine at pH 7.2. The concentrations of the two salts in the solution are shown in Tables 5.1.1 and 5.1.2. Each condition was performed in duplicate.
• the MAB-MYA load challenge in each of these wells was of 5.0 mg/mL of resin Table 5.1.1: Sodium chloride levels in each well (in mM)
• each well was equilibrated in the conditions of sodium chloride and sodium phosphate as described in Tables 5.1.1 and 5.1.2, in a phase volume ratio of 6:1 (30OuL solution: 5OuL resin). The plate was shaken for 20 minutes, allowing equilibrium to be reached. The solution was then removed by centrifuging the filter plate. This equilibration cycle was repeated three times.
• MAb-MYA solution to the appropriate protein load challenge with a volume ratio of 6: 1 (30OuL solution: 5OuL resin) at the appropriate sodium chloride and sodium phosphate concentration.
• the loaded plate was shaken for 20 minutes, allowing the resin and solution to equilibriate.
• the supernatant was removed from the filter plate by centrifugation and collected into a collection plate.
• the protein concentration in the supernatant in each well was determined by absorbance at A280nm.
• the Kp value can be used to describe regions where
• MAB-MYA binds to the hydroxyapatite medium with different strengths.
• the culture containing the monoclonal antibody was purified using a
• MabSelect column A Mabselect Protein A column was equilibrated with 5 column volumes of 50 mM Tris/150 mM NaCl, pH 7.5 at a flow rate of 300 cm/hr. The column was then loaded at a load of approximately 40 mg product/ml resin. This was followed by a lOCV wash in IM arginine, 5OmM Tris, pH 7.5 and a 5CV wash containing 10 niM Tris, 75mM NaCl, pH 7.5 wash. The column was then eluted using 10OmM arginine, 5OmM NaCl, pH 3.0. The product pool was neutralized to pH 7.2 using 2M HEPES pH 8.0.
• the partially purified antibody pools from the Protein A step were further purified over hydroxyapatite.
• the column diameter was 0.5 cm and the column height was 10 cm.
• Equilibration 1 300 mM sdium posphate, 1.0M NaCl, pH 6.8 (3 column volumes)
• Equilibration 2 5 - 30 mM sodium phosphate, 50 - 760 mM NaCl, 10OmM Arg, 10OmM HEPES pH 7.2 (5 column volumes) Wash 5 - 30 mM sodium phosphate, 50 - 760 mM NaCl, 10OmM Arg, 10OmM HEPES pH 7.2 (5 - 10 column volumes)
• a third mode of chromatography (hydroxyapatite) was shown to operate successfully in a weak partitioning mode. Protein A and HCP bind more tightly than the product antibody to ceramic resin, and are retained strongly under WP conditions. Higher values of Kp in the WP region are between 10 and 20 in some cases, which still provide good product recovery (> 90%). Lower levels of Kp give correspondingly higher recoveries.
• the partition coefficient in hydroxyapatite is a complex function of pH, salt (type and concentration), phosphate, and buffer components. All of these variables in general have an impact on the performance of the column step.
• the approach presented here provides a simple means of relating the impact of changing any one of these variables on column performance.
• the unified 'partition coefficient' approach presented in this example opens up the possibility of operating in a wider operating space in this mode of chromatography than has been done before.
• the weak partitioning conditions for optimum performance can easily be identified using the HTS methods described above.
• a high throughput screen was performed to identify the weak partitioning and flow-through conditions for Mab-A5T with ceramic hydroxyapatite medium. This screen varied pH, sodium chloride and sodium phosphate concentrations to determine their effect on the extent of binding of MAB-A5T to the hydroxyapatite medium.
• 50 ⁇ L of ceramic hydroxyapatite medium was added to 36 wells of a 96 well filter plate. Each well was equilibrated in solutions made up of the appropriate sodium chloride and sodium phosphate concentrations in a 5OmM HEPES buffer containing 5OmM arginine at either pH 7.0 or pH 8.0.
• the concentrations of the two salts in the solution are shown in Tables 6.1.1 and 6.1.2.
• the conditions shown in columns 1-3 were performed at pH 7.0, and columns 4-6 were performed at pH 8.0.
• the MAB-A5T load challenge in each of these wells was of 5.0 mg/mL of resin.
• each well was equilibrated in the conditions of sodium chloride, sodium phosphate and pH as described in Tables 6.1.1 and 6.1.2 in a phase volume ratio of 6:1 (30OuL solution: 5OuL resin). The plate was shaken for 20 minutes, allowing equilibrium to be reached. The solution was then removed by centrifuging the filter plate. This equilibration cycle was repeated three times.
• the resin in each well was challenged with a concentrated
• the loaded plate was shaken for 20 minutes, allowing the resin and solution to equilibrate.
• the supernatant was removed from the filter plate by centrifugation and collected into a collection plate.
• the protein concentration in the supernatant in each well was determined by absorbance at A280nm.
• resin was washed by adding solutions of the specified sodium chloride, sodium phosphate and pH conditions listed in Tables 6.1.1 and 6.1.2. The supernatant was removed after shaking for 20 minutes.
• a buffer comprising 10OmM sodium phosphate, IM NaCl pH 7.2 was added to remove the remaining protein that was bound to the resin.
• the partition coefficients were calculated for each well using the mass eluted from stage 4 and the product concentration from stage 2, and are shown in Table 6.1.3.
• the Kp value can be used to identify regions where
• MAB-A5T binds to the hydroxyapatite medium with different strengths.
• the strength of MAB-A5T binding to ceramic hydroxyapatite medium can be manipulated by varying conditions of NaCl, phosphate and pH into flow-through, weak partitioning, and binding zones.
• the culture containing the monoclonal antibody was purified using a
• MabSelect column A Mabselect Protein A column was equilibrated with 5 column volumes of 50 mM Tris/150 mM NaCl, pH 7.5 at a flow rate of 300 cm/hr. The column was then loaded at a load challenge of approximately 40 mg product/ml resin. This was followed by a 5CV wash in IM NaCl, 5OmM Tris, pH 7.5 and a 5CV wash containing 10 mM Tris, 75mM Nacl, pH 7.5 wash. The column was then eluted using 10OmM arginine, 5OmM NaCl, pH 3.0. The product pool was neutralized to pH 7.2 using 2M HEPES pH 8.0.
• the partially purified antibody pools from the Protein A step were further purified over hydroxyapatite.
• the column diameter was 0.5 cm and the column height was
• Table 6.2.1 Partition coefficients for MAB-A5T on cHA resin and the corresponding operating window.
• the operating conditions in these experiments correspond to the flow-through, weak partitioning (WP) region and binding regions.
• the HTS experiment described in Experiment 6.1 provides estimates for the value of the partition coefficient (Kp) under these conditions of pH, chloride and phosphate concentrations.
• the runs in Table 6.2.1 are ranked by the partition coefficients.
• the bound product was determined by measuring the protein in the column strip using UV absorbance. This method of determining the bound product typically underestimates the amount of product bound during the load due to the gradual desorption of the product during the wash. HCP and product related HMW removal, as well as product recovery results from these experiments are presented in Figure 12.
• a second example was presented in hydroxyapatite where operating under weak partitioning chromatography was shown to provide improved performance with respect to HCP and HMW reduction and product recovery (> 80%).
• the performance of the step was optimized primarily through the choice of partition coefficients used to run the column.
• the approach presented here provides a simple means of relating the impact of changing any one of several variables (pH, salt, phosphate, imidazole, glycine, HEPES, etc.,) to column performance.
• the weak partitioning conditions for optimum performance can easily be identified using the HTS methods described in this example.
• the approach presented here opens up the possibility of operating in a wider operating space in this mode of chromatography than has been done before.
• the optimal WP region in this example corresponds to partition coefficients between 2 and 20.
• a high throughput screen was performed to identify flow-through, weak partitioning and binding conditions for Mab-MYO with ceramic hydroxyapatite medium. This screen varied the concentration of pH, arginine/glycine, HEPES, sodium phosphate and sodium chloride to determine their effect on the extent of binding of MAB- MYO to the hydroxyapatite medium.
• the operating conditions in these experiments correspond to the flow-through, weak partitioning (WP) and binding regions.
• the HTS experiment described in Experiment 7.1 provides estimates for the value of the partition coefficient (Kp) under these conditions of pH, chloride, phosphate, glycine / arginine and HEPES concentration.
• the runs in Table 7.2.1 are ranked by the partition coefficients.
• the bound product was determined by measuring the protein in the column strip using UV absorbance. This method of determining the bound product typically underestimates the amount of product bound during the load due to the gradual desorption of the product during the wash.
• the product related High Molecular weight (HMW) removal and product recovery results from these experiments are also presented in Figure 13.
• a second example was presented in hydroxyapatite where operating under weak partitioning chromatography was shown to provide improved performance with respect to HMW reduction with good product recovery (> 80%).
• Product related HMW species and HMW species bind more tightly to ceramic resin than the product antibody, and is retained strongly under WP conditions.
• the WP region in this example corresponds to partition coefficients between 8 and 20.
• a culture containing a monoclonal antibody was purified using MabSelect resin.
• a Mabselect Protein A column was equilibrated with 5 column volumes of 50 mM Tris/150 mM NaCl, pH 7.5. The column was then loaded at a load of approximately 40 mg product/ml resin. This was followed by a 5CV wash in IM NaCl, 5OmM Tris, pH 7.5 and a 5CV wash containing 10 mM Tris, 75mM NaCl, pH 7.5 wash. The column was then eluted using 3OmM HEPES, pH 3.1. The product pool was neutralized to pH 7.2 using IM HEPES pH 8.0, resulting in a total HEPES concentration of 55mM. At pH 7.2, the HEPES contributes 17mM ionic strength to the buffer.

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